The faithful transmission of undamaged chromosomes to daughter cells at cell division is essential for life and for suppression of human disease. Genomic DNA is packaged into proteinaceous chromatin, upon which the crucial processes of transcription, DNA replication and repair occur. These processes are facilitated by the evolutionarily conserved SMC family, which includes cohesin (Smc1-Smc3), required for sister chromatid cohesion and centromere function;condensin (Smc2-Smc4), required with topoisomerase II to compact and drive the decatenation of chromosomes at mitosis;and the Smc5-Smc6 complex, whose essential function(s) are undefined. Our long-term objective is to dissect and define critical regulatory interfaces between the DNA repair, replication and cell cycle progression mechanisms. Ultimately, this will yield a molecular level understanding of the interplay between these processes that should explain the etiology of many human diseases and perhaps highlight potential therapeutic strategies. Currently, we are defining the functional interface between the Smc5-Smc6 complex and the replication monitoring checkpoint, enforced by Cds1 (hCHK2). Our studies utilize the genetically tractable and well-proven fission yeast model organism. For our Specific Aims below, fission yeast is ideal, as it has complex heterochromatic centromeres and associated factors that are well conserved in humans. Furthermore, components of the replication checkpoint are also highly conserved. We will characterize the Smc5-Smc6 holocomplex by integrating powerful yeast genetics with cutting-edge structural experiments, biochemistry and mass spectrometry. We have two Specific Aims in which we will determine the mitotic and meiotic chromosome segregation role(s) of Smc5-Smc6 and the associated DNA repair protein, Rad60. As Rad60 physically interacts with both Smc5-Smc6 and the replication checkpoint kinase Cds1, it provides an interface between the replication checkpoint and DNA repair processes. In our first Aim we will test the physiological importance of the Cds1-Rad60 interface in suppressing toxic replication-associated recombination. In addition, Rad60 family proteins contain the unique structural signature of tandem SUMO-like domains (SLDs). Therefore, we will functionally characterize Rad60 by solving the structures of its SLDs, which will facilitate mutagenesis coupled with in vivo phenotypic analyses. These mutagenesis studies will define the mechanism(s) by which Rad60 mediates its observed role in heterochromatin formation and stability. In our second Aim we will characterize the mitotic and meiotic homologous recombination repair and centromeric functions of the Smc5-6 complex. This will be greatly facilitated by identifying targets of the SUMO E3 ligase Smc5-Smc6 subunit Nse2. These studies will yield a detailed picture of the ways in which the evolutionarily conserved Smc5-Smc6, Rad60 and replication checkpoint maintain chromosome integrity and thus in humans, suppress disease priming genomic lesions. PUBLIC HEALTH RELEVANCE We have identified a molecular "machine" that protects and repairs the cells "blueprint" or genome. The genome contains information required to maintain normal cell growth and if this information is damaged, cancer can ensue. Our studies will provide key insights on normal regulation of cell growth and furthermore, reveal a potential "Achilles'Heel" of cancer cells.